An ability to detect the presence of a nucleic acid molecule having a particular predetermined sequence is of substantial importance in a variety of fields, such as forensics, medicine, epidemiology and public health, and in the prediction and diagnosis of disease. Such an ability can aid criminal investigations, by excluding wrongly accused individuals or by implicating culpable parties. It can be exploited to permit the identification of the causal agent of infectious disease, or the characterization of tumors and tissue samples, or ensure the wholesomeness of blood products.
An ability to detect the presence of a particular nucleic acid sequence in a sample is important in predicting the likelihood that two individuals are related to one another, or that an individual will suffer from a genetic disease. Such an ability can also be used in assays to determine the purity of drinking water, milk, or other foods.
In many cases of interest, the desired nucleic acid sequence is present at a very low concentration in the sample. In such cases, unless assay sensitivity can be increased through the use of sophisticated labels, the presence of the desired molecule may escape detection. Assay sensitivity may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose: enzyme labels (Kourilsky et al.; U.S. Pat. No. 4,581,333); radioisotopic labels (Falkow et al., U.S. Pat. No. 4,358,535; Berninger, U.S. Pat. No. 4,446,237); fluorescent labels (Albarella et al., EP 144914); chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al., EP 119448), etc.
Although the use of highly detectable labeled reagents can improve the sensitivity of nucleic acid detection assays, the sensitivity of such assays remains limited by practical problems which are largely related to non-specific reactions that increase the background signal produced in the absence of the nucleic acid the assay is designed to detect. Thus, for some applications, the anticipated concentration of the desired nucleic acid molecule will be too low to permit its detection by any of the above-described methods.
One method for overcoming the sensitivity limitation of nucleic acid concentration is to selectively amplify the nucleic acid molecule whose detection is desired prior to performing the assay. In vivo recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized (Cohen et al., U.S. Pat. No. 4,237,224; Sambrook, J. et al., In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment.
Recently, in vitro amplification methods have been developed. The impact of such methods has been phenomenal--without such amplification, most of the foregoing exemplary fields would not be possible. Thus, as the areas in which DNA amplification has expanded, the requirements placed upon various amplification techniques have changed. Accordingly, a very real and ongoing need exists for highly specific amplification techniques.
Perhaps the most widely practiced of these methods is the "polymerase chain reaction" ("PCR") (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194), which references are incorporated herein by reference).
PCR achieves the amplification of a specific nucleic acid sequence using two oligonucleotide primers complementary to regions of the sequence to be amplified. Extension products incorporating the primers then become templates for subsequent replication steps. The method selectively increases the concentration of a desired nucleic acid molecule even when that molecule has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single or double stranded DNA.
The method involves the use of a DNA polymerase to direct the template-dependent, extension of a pair of oligonucleotide primers. The primer extension products then become templates for subsequent replication steps.
The precise nature of the two oligonucleotide primers of the PCR method is critical to the success of the method. As is well known, a molecule of DNA or RNA possesses directionality, which is conferred through the 5'.fwdarw.3' linkage of the sugar-phosphate backbone of the molecule. Two DNA or RNA molecules may be linked together through the formation of a phosphodiester bond between the terminal 5' phosphate group of one molecule and the terminal 3' hydroxyl group of the second molecule. Polymerase dependent amplification of a nucleic acid molecule proceeds by the addition of a 5' nucleoside triphosphate to the 3' hydroxyl end of a nucleic acid molecule. Thus, the action of a polymerase extends the 3' terminus of a nucleic acid molecule. The oligonucleotide sequences of the two PCR primers are selected such that they contain sequences identical to, or complementary to, sequences which flank the sequence of the particular nucleic acid molecule whose amplification is desired. More specifically, the nucleotide sequence of the "first" primer is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3' to the sequence of the desired nucleic acid molecule, whereas the nucleotide sequence of the "second" primer is selected such that it contains a nucleotide sequence identical to one present 5' to the sequence of the desired nucleic acid molecule. Both primers possess the 3' hydroxyl groups which are necessary for enzyme mediated nucleic acid synthesis.
The PCR reaction is capable of exponential amplification of specific nucleic acid sequences because the extension product of the "first" primer contains a sequence which is complementary to a sequence of the "second" primer, and thus will serve as a template for the production of an extension product of the "second" primer. Similarly, the extension product of the "second" primer, of necessity, contain a sequence which is complementary to a sequence of the "first" primer, and thus will serve as a template for the production of an extension product of the "first" primer. Thus, by permitting cycles of hybridization, polymerization, and denaturation, a geometric increase in the concentration of the desired nucleic acid molecule can be achieved.
PCR technology is useful in that it can achieve the rapid and extensive amplification of a polynucleotide molecule (Mullis, K. B., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Saiki, R. K., et al., Bio/Technology 3:1008-1012 (1985); Mullis, K. B., et al., Met. Enzymol. 155:335-350 (1987), which references are incorporated herein by reference). Nevertheless, several practical problems exist with PCR. First extraneous sequences along the two templates can hybridize with the primers; this results in co-amplification due to such non-specific hybridization. As the level of amplification increases, the severity of such co-amplification also increases. Second, because of the ability of PCR to readily generate millions of copies for each initial template, accidental introduction of the end-product of a previous reaction into other samples easily leads to false-positive results. Third, PCR, does not, in and of itself, allow for detection of single-base changes, i.e. the protocol does not intrinsically discriminate between a "normal" sequence and an allelic variant sequence.
The advent of PCR led to the development of additional amplification methods. One such alternative method is the "Ligase Chain Reaction" ("LCR") (Barany, F., Proc. Natl. Acad. Sci. (U.S.A.) 88:189-193 (1991). LCR uses two pairs of oligonucleotide probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependent ligase. Thus, the hybridization of the first pair of oligonucleotides to a "first" strand of the target, permits the oligonucleotides to be ligated together. The sequence of the second pair of oligonucleotides is selected such that the oligonucleotides can hybridize to abutting sequences of this ligation product, thereby forming a second substrate for ligation. The ligation product of the second strand thus possesses a sequence that is substantially identical to that of the "first" strand of the target.
As with PCR, the resulting products thus serve as templates in subsequent cycles and an exponential amplification of the desired sequence is obtained. Beneficially, LCR can be utilized to detect mutations, and in particular, single nucleotide mutations. Thus, the primers can be designed such that they can be ligated together only if the target molecule either contains or lacks a predetermined mutational site.
One problem associated with LCR is that, by definition, the procedure requires four oligonucleotides and a ligase, and may result in the non-specific "blunt-end ligation" of the oligonucleotides. Such non-specific "blunt-end ligation," if it occurs, will cause a target-independent exponential amplification of the oligonucleotides. This can lead to high background signal or false-positive results.
This deficiency can, in some respects, be addressed using oligonucleotides that hybridize to adjacent, but non-abutting sequences (PCT Appl. WO 90/01069). As in LCR, such a method involves the use of two sets of primers. However, since the primers are designed to hybridize to non-abutting sequences of the target molecule, the hybridization product contains a "gap" separating the hybridized oligonucleotides. These gaps are then "filled" with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus, at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential amplification of the desired sequence is obtained.
While this protocol avoids the LCR problem of non-specific blunt end ligation in the absence of target, it does so at the expense of LCR's capacity to detect single base mutational changes, and requires that the sequence of the entire "gap" be known in advance. In addition, a critical difficulty in using this technique is the need to design the oligonucleotide primers such that the "gap" can be "repaired" with only a subset of the dNTPs. I.e., the gap cannot comprise all four of the bases such that only a maximum of three of the four dNTPs can be added to the reaction vessel.
The "Oligonucleotide Ligation Assay" ("OLA") (Landegren, U. et al., Science 241:1077-1080 (1988)) shares certain similarities with LCR. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. OLA, like LCR, is particularly suited for the detection of point mutations. Unlike LCR, however, OLA results in "linear" rather than exponential amplification of the target sequence. A problem associated with OLA, then, is the lack of exponential amplification.
Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. In addition to requiring multiple, and separate, processing steps, one problem associated with such combinations is that they inherit all of the problems associated with PCR and OLA.
Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh D et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras, T. R. et al., PCT appl. WO 88/10315 (priority: U.S. patent applications Ser. Nos. 064,141 and 202,978)). Schemes based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, are also known (Wu, D. Y. et al., Genomics 4:560 (1989)).
Miller, H. I. et al., PCT appl. WO 89/06700 (priority: U.S. patent application Ser. No. 146,462, filed Jan. 21, 1988), disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme was not cyclic; i.e. new templates were not produced from the resultant RNA transcripts.
Malek, L. T. et al., U.S. Pat. No. 5,130,238, and Davey, C. et al. (European Patent Application Publication no. 329,822) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA). The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5'-to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. An improvement of this method was developed by Schuster et al. (U.S. Pat. No. 5,169,766) who show that the primer extension taught by Malek (U.S. Pat. No. 5,130,238) is not necessary.
All of the above amplification procedures depend on the principle that an end product of a cycle is functionally identical to a starting material. Thus, by repeating cycles, the nucleic acid is amplified exponentially.
An isothermal amplification method has been described in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[.alpha.-thio]triphosphates in one strand of a restriction site (Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)).
Methods that use thermo-cycling, e.g. PCR or Wu, D. Y. et al., Genomics 4:560 (1989)), have a theoretical maximum increase of product of 2-fold per cycle, because in each cycle a single product is made from each template. In practice, the increase is always lower than 2-fold. Further slowing the amplification is the time spent in changing the temperature. Also adding delay is the need to allow enough time in a cycle for all molecules to have finished a step. Molecules that finish a step quickly must "wait" for their slower counterparts to finish before proceeding to the next step in the cycle; to shorten the cycle time would lead to skipping of one cycle by the "slower" molecules, leading to a lower exponent of amplification.
Methods that include a transcription step, e.g. that of the present invention or of Malek, L. T. et al. (U.S. Pat. No. 5,130,238) or Davey, C. et al. (European Patent Application Publication no. 329,822), can increase product by more than a factor of 2 at each cycle. Indeed, as 100 or more transcripts can be made from a single template, factors of increase of 100 or more are theoretically readily attainable. Furthermore, if all steps are performed under identical conditions, no molecule which has finished a particular step need "wait" before proceeding to the next step. Thus amplifications that are based on transcription and that do not require thermo-cycling are potentially much faster than thermo-cycling amplifications such as PCR.
In sum, although a variety of amplification methods have been developed, a strictly target-dependent method that is capable of mediating the exponential amplification of a target molecule, and which possesses the ability to detect single nucleotide allelic variation would be highly desirable. The present invention provides such a method.